U.S. patent application number 11/612587 was filed with the patent office on 2007-06-28 for method for detecting low concentrations of a target bacterium that uses phages to infect target bacterial cells.
This patent application is currently assigned to COLORADO SCHOOL OF MINES. Invention is credited to Angelo J. Madonna, Jon C. Rees, Kent J. Voorhees.
Application Number | 20070148638 11/612587 |
Document ID | / |
Family ID | 29250416 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070148638 |
Kind Code |
A1 |
Madonna; Angelo J. ; et
al. |
June 28, 2007 |
Method for Detecting Low Concentrations of a Target Bacterium That
Uses Phages to Infect Target Bacterial Cells
Abstract
The invention is directed to a method for detecting low
concentrations of bacteria in liquid solution that may or may not
be complex liquid solutions. In one embodiment, immunomagnetic
separation (IMS) is used to separate target bacterium that may be
in a liquid mixture from other constituents in the mixture. A low
concentration of a bacteriophage for the target bacteria is
subsequently used to infect target bacterial cells that have been
captured using the IMS technique. If at least a certain
concentration of target bacterium are present, the bacteriophage
will multiply to a point that is detectable. Matrix assisted laser
desorption ionization/time-of-flight-mass spectrometry
(MALDI/TOF-MS) is then used to produce a mass spectrum that is
analyzed to determine if one or more proteins associated with the
bacteriophage are present, thereby indirectly indicating that
target bacterium were present in the liquid mixture.
Inventors: |
Madonna; Angelo J.; (Tooele,
UT) ; Voorhees; Kent J.; (Golden, CO) ; Rees;
Jon C.; (Golden, CO) |
Correspondence
Address: |
CHRISTOPHER J. KULISH, ESQ
HOLLAND & HART LLP
P. O. BOX 8749
DENVER
CO
80201-8749
US
|
Assignee: |
COLORADO SCHOOL OF MINES
1500 Illinois Street
Golden
CO
80401
|
Family ID: |
29250416 |
Appl. No.: |
11/612587 |
Filed: |
December 19, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10249452 |
Apr 10, 2003 |
7166425 |
|
|
11612587 |
Dec 19, 2006 |
|
|
|
60319184 |
Apr 12, 2002 |
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Current U.S.
Class: |
435/5 ;
435/7.32 |
Current CPC
Class: |
G01N 33/56911 20130101;
G01N 33/54326 20130101; C12Q 1/04 20130101 |
Class at
Publication: |
435/005 ;
435/007.32 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; G01N 33/554 20060101 G01N033/554 |
Claims
1. A method for determining if a target bacterium is present in a
liquid solution when the target bacterium is or may be present in a
low concentration that is near or below the detection limit for a
particular detection technology comprising: making a determination
as to whether a low concentration of a target bacterium may be
present in a liquid solution; using, following said step of making
and if a determination is made that a low concentration of said
target bacterium may be present, a first quantity of a
bacteriophage to infect at least some of any target bacterium that
are present in said liquid solution and multiply the number of
bacteriophage in said liquid solution; and analyzing, following
said step of using, at least a portion of said liquid solution to
determine if a biomarker for said bacteriophage is present that
indirectly indicates that said target bacterium is also present in
said liquid solution.
2. A method, as claimed in claim 1, wherein: said step of making
comprises assuming that any of said target bacterium that are
present in said liquid solution are present in said liquid solution
in a low concentration.
3. A method, as claimed in claim 1, wherein: said step of making
comprises performing an assay to determine if a biomarker for said
target bacterium is present that is indicative of the presence of a
concentration of said target bacterium that reliably exceeds the
detection limit of the detection technology.
4. A method, as claimed in claim 3, wherein: said step of
performing an assay comprises performing a mass spectrum
analysis.
5. A method, as claimed in claim 1, wherein: said step of using
comprises applying a first quantity of said bacteriophage to said
liquid solution such that there is likely to be a high multiplicity
of infection ("MOI") number.
6. A method, as claimed in claim 1, wherein: said step of analyzing
comprises performing a mass spectrum analysis.
7. A method, as claimed in claim 1, wherein: said step of analyzing
comprises performing a MALDI analysis.
8. A method, as claimed in claim 1, wherein: said step of analyzing
comprises performing a MALDI-TOF analysis.
9. A method, as claimed in claim 1, wherein: said step of analyzing
comprises performing an electro-spray ionization mass spectrometry
analysis.
10. A method, as claimed in claim 1, wherein: said step of
analyzing comprises performing an ion mobility spectrometry
analysis.
11. A method, as claimed in claim 1, wherein: said step of
analyzing comprises performing an optical spectroscopy
analysis.
12. A method, as claimed in claim 1, wherein: said step of
analyzing comprises performing an immuno analysis.
13. A method, as claimed in claim 1, wherein: said step of
analyzing comprises performing a chromatographic analysis.
14. A method, as claimed in claim 1, wherein: said step of
analyzing comprises performing an aptamer analysis.
15. A method, as claimed in claim 1, further comprising:
determining whether said liquid solution is likely to contain a
biological element with a biomarker that could create a false
positive for a biomarker for said bacteriophage.
16. A method, as claimed in claim 15, wherein: said step of
determining comprises assuming that a biological element is present
in said liquid solution that has a biomarker that could create a
false positive for a biomarker for said bacteriophage.
17. A method, as claimed in claim 15, wherein: said step of
determining comprises performing an assay to determine if a
biomarker is present that could create a false positive for a
biomarker for said bacteriophage.
18. A method, as claimed in claim 15, further comprising: after a
determination has been made that said biological element may
present, separating said target bacterium from said liquid
solution.
19. A method, as claimed in claim 18, wherein: said step of
separating comprises using immuno-magnet beads coated with
antibodies for said target bacteria.
20. A method, as claimed in claim 18, further comprising: after a
determination has been made said step of separating is performed
prior to said step of using.
21. A method, as claimed in claim 18, wherein: after a
determination has been made that said biological element may
present and after said step of using, separating said bacteriophage
from said liquid solution.
22. A method for determining if a target bacterium is present in a
liquid solution when the target bacterium is or may be present in a
low concentration that is near or below the detection limit for a
particular detection technology comprising: making a determination
as to whether a low concentration of a target bacterium may be
present in a liquid solution; using, following said step of making
and if a determination is made that a low concentration of said
target bacterium may be present, a first quantity of a biotinylated
bacteriophage to infect at least some of any target bacterium that
are present in said liquid solution and multiply the number of
bacteriophage in said liquid solution; and analyzing, following
said step of using, said liquid solution to determine if a
biomarker for said bacteriophage is present that indirectly
indicates that said target bacterium is also present in said liquid
solution.
23. A method, as claimed in claim 22, wherein: said step of using
comprises applying a first quantity of said biotinylated
bacteriophage to said liquid solution such that there is likely to
be a high multiplicity of infection ("MOI") number.
24. A method, as claimed in claim 22, wherein: said step of using
comprises applying a first quantity of said biotinylated
bacteriophage to said liquid solution that is near or likely to
exceed the multiplicity of infection ("MOI") number, wherein said
first quantity of biotinylated bacteriophage are attached to a
strepavidin coated probe.
25. A method, as claimed in claim 22, wherein: said step of
analyzing comprises separating a substantial portion of said
biotinylated bacteriophage from said liquid solution to produce a
remaining liquid solution.
26. A method, as claimed in claim 25, wherein: said step of
separating comprises using a strepavidin coated probe.
27. A method, as claimed in claim 26, wherein: said step of using a
strepavidin coated probe comprises separating a strepavidin coated
probe from said liquid solution, said strepavidin coated probe
having been used to apply said biotinylated bacteriophage to said
liquid solution in said step of using.
28. A method, as claimed in claim 26, wherein: said step of using a
strepavidin coated probe comprises applying a strepavidin coated
probe to said liquid solution to capture biotinylated bacteriophage
in said liquid solution.
29. A method, as claimed in claim 25, wherein: said step of
analyzing comprises assaying said remaining liquid solution to
determine if a biomarker for said bacteriophage is present.
30. A method, as claimed in claim 29, wherein: said step of
assaying comprises performing a mass spectrum analysis.
31. A method, as claimed in claim 22, further comprises: separating
said target bacterium from said mixture.
32. A method, as claimed in claim 31, wherein: said step of
separating is performed prior to said step of using.
33. A method, as claimed in claim 22, further comprises: separating
said biotinylated bacteriophage from said mixture.
34. A method, as claimed in claim 33, wherein: said step of
separating is performed after said step of using.
35. A method for determining if a target bacterium is present in a
liquid solution when the target bacterium is or may be present in a
low concentration that is near or below the detection limit for a
particular detection technology comprising: first determining
whether a low concentration of a target bacterium may be present in
a liquid solution; second determining whether a biological element
may be present in said liquid solution that has a biomarker that
could create a false positive for a biomarker for a bacteriophage
used in said subsequent step of using and used in said step of
analyzing as an indirect biomarker for the presence of said target
bacterium; first using, following said step of first determining
and when a determination has been made that a low concentration of
said target bacterium may be present, a first quantity of a
biotinylated bacteriophage to infect at least some of any target
bacterium that are present in said liquid solution and multiply the
number of bacteriophage in said liquid solution; second using,
following said step of second determining and when a determination
has been made that a biological element may be present,
purification technique; and analyzing, following said steps of
first using and second using, said liquid solution to determine if
a biomarker for said bacteriophage is present that indirectly
indicates that said target bacterium is also present in said liquid
solution.
36. A method, as claimed in claim 35, wherein: said step of second
using comprises performing a purification technique that separates
at least some of any target bacterium that are present in said
liquid solution from said liquid solution.
37. A method, as claimed in claim 35, wherein: said step of
performing occurs before said step of first using.
38. A method, as claimed in claim 37, wherein: said step of
performing comprises using immuno-magnetic beads to which are
attached antibodies for said target bacterium.
39. A method, as claimed in claim 35, wherein: said step of second
using comprises performing a purification technique that separates
said bacteriophage from said liquid mixture.
40. A method, as claimed in claim 39, wherein: said step of
performing occurs after said step of first using.
41. A method, as claimed in claim 39, wherein: said step of
performing comprises using immuno-magnetic beads to which are
attached antibodies for said bacteriophage.
42. A method for determining if a target bacterium is present in a
liquid solution when the target bacterium is or may be present in a
low concentration that is near or below the detection limit for a
particular detection technology comprising: making a determination
as to whether a low concentration of a target bacterium may be
present in a liquid solution; using, following said step of making
and if a determination is made that a low concentration of said
target bacterium may be present, a first quantity of a tagged
bacteriophage to infect at least some of any target bacterium that
are present in said liquid solution and produce a number of
untagged bacteriophage in said liquid solution; and analyzing said
liquid solution to determine if a biomarker for said untagged
bacteriophage and that is distinguishable relative to any
biomarkers for said tagged bacteriophage is present and that
indirectly indicates that said target bacterium is also present in
said liquid solution.
43. A method, as claimed in claim 42, wherein: said step of
analyzing comprises separating said tagged bacteriophage from said
liquid solution.
44. A method, as claimed in claim 42, wherein: said step of
analyzing comprises using a mass spectrometry analysis technique to
produce a mass spectrum.
45. A method, as claimed in claim 44, wherein: said step of using
comprises inspecting said mass spectrum to determine if a biomarker
that is unique to said untagged bacteriophage relative to said
tagged bacteriophage is present.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/319,184, entitled "METHOD OF DETECTING LOW
CONCENTRATIONS OF A TARGET BACTERIA THAT USES PHAGES TO INFECT
TARGET BACTERIAL CELLS" and filed by Angelo J. Madonna and Kent J.
Voorhees on Apr. 12, 2002, and U.S. Non-provisional application
Ser. No. 10/249,452, entitled "METHOD OF DETECTING LOW
CONCENTRATIONS OF A TARGET BACTERIA THAT USES PHAGES TO INFECT
TARGET BACTERIAL CELLS" and filed by Angelo J. Madonna and Kent J.
Voorhees on Apr. 10, 2003, which applications are incorporated by
reference into this application in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for detecting low
concentrations of a target bacterium in a liquid mixture that uses
bacteriophages to infect target bacterial cells.
BACKGROUND OF THE INVENTION
[0003] Standard microbiological methods have relied on
substrate-based assays to test for the presence of specific
organisms (Bordner, et al. 1978). These techniques offer very high
levels of selectivity but are hindered by the requirement to first
grow or cultivate pure cultures of the targeted organism, which can
take 24 hours or longer. This time constraint severely limits the
effectiveness to provide a rapid response to the presence of
virulent strains of microorganisms.
[0004] Molecular biology techniques are quickly gaining acceptance
as valuable alternatives to standard microbiological tests. In
particular, serological methods have been widely employed to
evaluate a host of matrices for targeted microorganisms (Kingsbury
& Falkow 1985; Wyatt et al. 1992). These tests focus on using
antibodies to first trap and then separate targeted organisms from
other constituents in complicated biological mixtures. Once
isolated, the captured organism can be concentrated and detected by
a variety of different techniques that do not require cultivating
the biological analyte.
[0005] One very popular approach, termed immunomagnetic separation
(IMS), involves immobilizing antibodies to spherical, micro-sized
paramagnetic beads and using the antibody-coated beads to trap
targeted microorganisms from liquid media. The beads are easily
manipulated under the influence of a magnetic field facilitating
the retrieval and concentration of targeted organisms. Moreover,
the small size and shape of the beads allow them to become evenly
dispersed in the sample, accelerating the rate of interaction
between bead and target. These favorable characteristics lead to
reductions in assay time and help streamline the analytical
procedure making it more applicable for higher sample throughput
and automation.
[0006] Downstream detection methods previously used with IMS
include ELISA (Cudjoe, et al. 1995), dot blot assay (Skjerve et al.
1990), electrochemiluminescence (Yu and Bruno 1996), and flow
cytometry (Pyle, et al. 1999). Although these tests provide
satisfactory results, they are laborious to perform and give binary
responses (yes/no) that are highly susceptible to false-positive
results due to cross-reactivity with non-target analytes. Recently
reported is a rapid method for identifying specific bacteria from
complex biological mixtures using IMS coupled to matrix-assisted
laser desorption/ionization (MALDI) time-of-flight (TOF) mass
spectrometry (MS)(Madonna et al. 2001). This approach allowed a
variety of matrices to be evaluated for the presence of a
Salmonella species within a total analysis time of 1 hour.
Moreover, the developed procedure required little sample
processing, was relatively easy to perform, and the molecular
weight information obtained made it possible to discriminate
between signals from the target bacteria and signals from
cross-reacted constituents.
[0007] MALDI-TOF-MS is a proven technique for identifying whole
cellular microorganisms (Holland et al (1996); van Barr 2000;
Madonna et al. 2000). In principle, MALDI is a `fingerprinting`
technique where mass spectra featuring varying distributions of
protein signals are generated. The signature profiles that are
produced, due to inherent differences in microbial proteomes, make
it possible to discriminate between organisms down to the strain
level (Arnold and Reilly 1998). The MALDI-TOF technique coupled
with IMS includes, in one embodiment, mixing immunomagnetic beads
specific to the target bacteria with the liquid mixture that may
contain the target bacteria for a short incubation period (e.g., 20
min). Any target bacteria captured by the beads are washed twice,
re-suspended in deionized H.sub.2O, and directly applied onto a
MALDI sample probe. The target bacteria-bead complex is then
overlaid with a micro-volume of matrix solution and dried at room
temperature. Irradiation of the resulting crystalline mass with a
high intensity laser promotes the liberation and ionization of
intact cellular proteins that are subsequently detected by a TOF
mass spectrometer. The resulting mass spectrum is then interrogated
for definitive mass peaks that signify the presence of the target
bacteria.
SUMMARY OF THE INVENTION
[0008] The invention is directed to a method for determining if a
target bacterium is present in a liquid solution when the target
bacterium is or may be present in a low concentration that is at or
near the detection limit for a particular detection technology. As
used herein, the term "target bacterium" refers to a specie of
species of bacteria. In turn, the invention is applicable to
situations in which it is desirable to determine whether a target
bacterium (e.g., E. coli) is present in a liquid solution when the
number of target bacterium per unit volume of solution (i.e., the
concentration of the target bacterium) is or may be below the
detection limit for a particular detection technology. In some
instances, a plurality of target bacterium may be referred to as
the target bacteria.
[0009] In one embodiment, the process comprises using a biological
amplification procedure in which bacteriophages for the target
bacterium are applied to the liquid solution. (Bacteriophages are
viruses that infect bacteria and in the process produce many
progeny. Structurally, the bacteriophage consists of a protein
shell (capsid) that encapsulates the viral nucleic acid. The capsid
is constructed from repeating copies of the same protein.
Bacteriophages are able to infect specific bacterial cells and
because of the multiplication of the genetic material, the cells
eventually burst releasing millions of copies of the original
phage.) The bacteriophages and any of the target bacterium present
in the liquid solution are allowed to incubate. During the
incubation period, the bacteriophages will multiply by infecting
target bacterium present in the solution. More specifically, the
bacteriophage replicates numerous copies of itself in an infected
target bacterium. Eventually, the infected target bacterium lyses
and the replicated or progeny bacteriophages are released into the
liquid solution. The solution is then analyzed to determine if a
biomarker for the bacteriophage is present, thereby indirectly
indicating that the target bacterium is present in the liquid
solution. Possible analysis techniques comprise mass spectrometry
techniques, such as MALDI-MS and electro-spray ionization-MS
techniques.
[0010] To assure that the detection of a biomarker for the
bacteriophage indicates that the target bacterium is present in the
liquid solution, a concentration of the bacteriophage is applied to
the liquid solution that is below the detection limit for the
biomarker for the bacteriophage for whatever analysis technique is
employed. This assures that if the biomarker for the bacteriophage
is detected by the analysis technique, the detectable concentration
of the biomarker is attributable to the replication of the
bacteriophage by the target bacterium present in the liquid
solution. In certain situations, the use of such a concentration of
bacteriophage has a multiplicity of infection ("MOI") (i.e., ratio
of infecting bacteriophages to target bacterium) that is too low to
rapidly produce a sufficient concentration of bacteriophages or
biomarkers for the bacteriophage for detection.
[0011] Another embodiment of the process addresses this problem by
adding a very high concentration of the bacteriophage to the liquid
solution, thereby assuring a high MOI. In this case, the
concentration of the bacteriophage added to the solution may exceed
the detection limit of whatever analysis technique is employed to
detect the bacteriophage or biomarker of the bacteriophage.
Consequently, the process applies parent bacteriophage to the
solution that can distinguished from any progeny bacteriophage
resulting from the infection of target bacterium in the mixture. If
the distinguishable progency bacteriophage or a distinguishable
biomarker of the progeny bacteriophage are present, this indicates
that the target bacterium is present in the solution.
[0012] In one embodiment, the parent bacteriophage (i.e., the
bacteriophage initially applied to the solution) are "tagged" so
that whatever analysis technique is employed is inherently capable
of distinguishing the parent bacteriophage or parent bacteriophage
biomarkers from the progeny bacteriophage or biomarkers for the
progency bacteriophage. For example, if a mass spectral analysis
technique is employed, the parent bacteriophage are "tagged" with a
substance that alters or shifts the mass spectrum of the parent
bacteriophage relative to the progeny bacteriophage, which will not
inherit the "tag" from the parent bacteriophage. For example, a
biotinylated bacteriophage can be employed as a parent
bacteriophage and will have a different mass spectrum than the
progeny bacteriophage produced by the biotinylated bacteriophage
infecting target bacterium present in the solution. Other "tags"
can be employed for other types of analytical techniques.
[0013] In another embodiment, the parent bacteriophage possesses a
characteristic that allows the parent bacteriophage to be separated
from the progeny bacteriophage in the liquid solution prior to
analysis, thereby assuring that most, if not all of the
bacteriophages present in the liquid solution after separation are
progeny bacteriophage resulting from the replication of the parent
bacteriophage by target bacteria present in the liquid solution. In
one embodiment, the parent bacteriophages initially applied to the
liquid solution are biotinylated bacteriophages. Biotinylated
bacteriophages are highly attracted to strepavidin. This attraction
is exploited to separate the biotinylated bacteriophage from
progeny bacteriophage resulting from replication of the
biotinylated bacteriophage by target bacterium present in the
mixture.
[0014] In one embodiment, the biotinylated bacteriophage are
attached to a strepavidin coated probe. Consequently, separation of
the biotinylated bacteriophage from the liquid solution after the
incubation period is accomplished by removing the probe from the
liquid solution. In another embodiment, strepavidin-coated magnetic
beads are applied to the liquid solution. The beads are used to
pick up the biotinylated bacteriophage. The beads are then
separated from the liquid solution using a magnet. In yet another,
embodiment a strepavidin coated probe (e.g., a slide) is applied to
the liquid solution after the incubation period. The biotinylated
bacteriophage adhere to the probe and then the probe is separated
from the liquid solution.
[0015] Yet a further embodiment of the invention recognizes that
the liquid solution in which the target bacterium may be present is
or may be a complex mixture that includes biological material that
makes the detection of the bacteriophage or biomarker for the
bacteriophage more difficult or reduces the reliability of the
information provided by the detection technology employed. For
instance, when a mass spectrometry detection methodology is
employed, the complex mixture may produce a signal in which the
biomarker associated with the bacteriophage is obscured or, stated
differently, has a low signal-to-noise ratio. To address this
possibility, the liquid solution is subject to a purification step
in which target bacterium that are present in the liquid solution
are separated from the remainder of the solution. In one
embodiment, immuno-magnetic separation ("IMS") is utilized to
separate target bacterium present in the liquid solution from the
remainder of the solution. In one particular embodiment, magnetic
beads are coated with an antibody for the target bacterium. The
antibodies pick up the target bacterium present in the liquid
mixture and then a magnet is used to separate the beads from the
remainder of the liquid solution. The beads and any adhering target
bacterium are then subjected to the biological amplification
process and analysis. It should be appreciated this purification
step also addresses the possibility that feral versions of the
bacteriophage may be present in the liquid solution and that such
versions could produce a false positive if the liquid solution was
not subjected to a purification step.
[0016] If feral versions of the bacteriophage are not of concern,
the purification step can be implemented after the biological
amplification process. In this embodiment, the purification step
involves separating the bacteriophages and the liquid solution,
rather than separating the target bacterium and the liquid
solution. In one embodiment, an IMS is used in which magnetic beads
are coated with an antibody for the bacteriophage. The beads pick
up the bacteriophages present in the solution and then a magnet is
used to separate the beads from the remainder of the solution.
[0017] In another embodiment of the invention, the analysis step
comprises using MS/MS analysis to determine if a biomarker for the
target bacterium is present. The use of MS/MS analysis produces a
highly reliable indication of the presence of a biomarker for a
target bacterium. As a consequence, at least in some cases, the use
of MS/MS analysis renders the need for a purification step
unnecessary.
[0018] In yet another embodiment, the invention is directed to a
process for detecting low concentrations of a target bacterium in
complex mixtures. In one embodiment, the process comprises using an
IMS procedure to isolate at least some of a target bacterium that
may be present in a liquid mixture. The process further includes
employing a biological amplification procedure in which a low titer
or concentration of bacteriophages for the target bacterium are
applied to at least some of the target bacterium that has been
isolated by the IMS procedure. The mixture of bacteriophages and
any of the target bacterium that has been isolated is allowed to
incubate. If at least a certain concentration of the target
bacterium is present, the bacteriophages will multiply during the
incubation period such that a high titer or concentration of
bacteriophages will be present in the mixture and detectable by
MALDI-TOF-MS analysis. If no or only a small number of the target
bacterium is present, there will be a low concentration of
bacteriophages present in the mixture that will not be reasonably
detectable by MALDI-TOF-MS analysis. Following incubation, a
MALDI-TOF-MS analysis is performed on the incubated mixture of
bacteriophages and target bacterium. The resulting mass spectrum is
analyzed to determine if a protein that is associated with the
bacteriophages is present. If the protein for the bacteriophage is
detected, then it can be concluded that at least a low
concentration of the target bacterium is present in the
mixture.
[0019] It should also be appreciated that the method of the
invention is capable of detecting low concentration of a target
bacterium regardless of the manner in which the bacterium was grown
or propagated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 depicts, for one embodiment of the invention, an
immunomagnetic bead that is used to isolate the target
microorganism (antibodies not drawn to scale);
[0021] FIG. 2 is a schematic representation of the immunomagnetic
purification step used to isolate a target antigen (Escherichia
coli) in one embodiment of the invention;
[0022] FIG. 3 is a schematic representation of the bacteriophage
amplification step for one embodiment of the invention;
[0023] FIG. 4 shows a typical MALDI-TOF mass spectrum obtained from
a high titer sample of MS2 in PBS; and
[0024] FIGS. 5A-5C illustrate how an embodiment of the invention
was able detect a biomarker of the bacteriophage indicative of
presence of E. coli for decreasing concentrations of E. coli.
DETAILED DESCRIPTION
[0025] Generally, the invention relates to the use of a
bacteriophage to indirectly detect the presence of a target
bacterium in a liquid solution where the concentration of the
target bacterium is or is likely to be near or below the detection
limit for the particular detection technology employed.
[0026] Bacteriophages are viruses that infect bacteria and in the
process of infecting the bacteria produce many progeny.
Structurally, the bacteriophage consists of a protein shell
(capsid) that encapsulates the viral nucleic acid. The capsid is
constructed from repeating copies of the same protein(s).
Bacteriophages are able to infect specific bacterial cells and
because of the multiplication of the number of progeny, the cells
eventually burst releasing millions of copies of the original
phage. This infection process has been utilized to serve as a
biomarker amplification step for detecting low concentrations of
target bacterial cells. For example, the capsid of the MS2
bacteriophage contains 180 copies of a 13 kDa protein. This
particular virus specifically infects strains of Escherichia coli
and is able to produce between 10,000 to 20,000 copies of itself
within 40 min after attachment to the target bacterial cell.
Essentially, one E. coli could be infected with MS2 resulting in
the replication of the capsid protein(s) by a factor of
1.8.times.10.sup.6.
[0027] The results from matrix assisted laser desorption
ionization/mass spectrometry (MALDI/TOF) can be used to show the
utility of the amplification step. MALDI-TOF-MS is a proven
technique for identifying whole cellular microorganisms (Holland et
al 1996; van Barr 2000; Madonna et al. 2000). In principle, MALDI
is a `fingerprinting` technique where mass spectra featuring
varying distributions of protein signals are generated. The
signature profiles are produced due to inherent differences in
microbial proteomes that make it possible to discriminate between
organisms down to the strain level (Arnold and Reilly 1998).
[0028] In an experiment where the protein MALDI signal from target
bacterial cells was too weak for detection, the addition of low
levels (too low to detect by MALDI) of the appropriate phage to the
target bacterial cells after about thirty minutes produced a
detectable protein MALDI signal attributable to the phage capsid
protein. Bacteriophages specific for other bacterial species
typically have capsid proteins of different molecular weight and
therefore give a different MALDI signal. Therefore, the procedure
is applicable to a multitude of different bacterial species. Other
detection technologies, such as ion mobility spectrometry, optical
spectroscopy, immuno techniques, chromatographic techniques and
aptamer processes, are also feasible.
[0029] Generally, the process for detecting low concentrations of a
target bacterium in a complex liquid mixture that contains or is
likely to contain biological material other than the target
bacterium comprises processing the mixture or a portion thereof to
produce a liquid mixture, solution or sample for analysis that, if
at least a certain concentration of the target bacterium is present
in the mixture, a discernable signal or indication thereof is
produced. It should be understood that the terms "liquid solution"
and "liquid mixture" refer to the original solution or mixture that
is the subject of the test and any liquid solutions or mixtures
that, as a result of the application of the method, contain a
portion of the original solution or mixture.
[0030] In one embodiment, the process comprises making a
determination if a low concentration of a target bacterium is
present in the liquid solution. This determination can be made by
assuming that any of the target bacterium that are present in the
liquid solution are present in a low concentration. Alternatively,
an assay can be performed to determine if the target bacterium is
present in a concentration that reliably exceeds the detection
limit of whatever detection technology is being utilized. For
instance, a mass spectrometry technique can be utilized. If the
mass spectrometry technique provides a reliable signal indicative
of the presence of the target bacterium in the liquid solution, no
further steps needs to be taken. If, however, the mass spectrometry
technique does not provide a reliable signal indicative of the
presence of the target bacterium in the liquid solution, then it
can be concluded that the target bacterium may be present in the
liquid solution but in a concentration that below or near the
detection limit of the mass spectrometry technique. In this case,
further steps are taken to determine whether the target bacterium
is present in the liquid solution in a concentration that is under
the detection limit of the mass spectrometry technique.
[0031] The process involves a purification step that involves
capturing the target bacterium that may be present in the mixture
and separating any of the captured bacterium from other biological
material that may be present in the mixture. By separating any of
the captured target bacterium from other biological material that
may be present in the mixture, the portion of the subsequently
produced mass spectrum signal associated with other biological
material present in the mixture is reduced. In one embodiment, an
immunomagnetic separation (IMS) technique is used to capture and
separate the target bacterium.
[0032] The process further comprises subjecting at least some of
any of the captured and separated target bacterium to an
amplification step in which the target bacterium are infected with
a bacteriophage that is specific to the target bacterium. If there
is at least a certain concentration of the target bacterium
present, the bacteriophage will multiply to a point that a
biomarker associated with the bacteriophage will be detectable
using an analysis technique, such as MALDI/TOF-MS. In the case of
MALDI/TOF-MS, a portion of the subsequently produced mass spectral
signal indicative of the presence of the bacteriophage will be
increased. If there is less than a particular concentration of the
target bacterium present, the bacteriophage will not multiply
sufficiently to be detectable in the mass spectrum produced using
MALDI/TOF-MS. In essence, provided there is a least a certain
concentration of the target bacterium present in the mixture, the
purification and amplification steps serve to increase the
signal-to-noise ratio for the portion of the subsequently produced
mass spectrum that is associated with the bacteriophage.
[0033] After amplification, at least a portion of the amplified
mixture is subjected to analysis to determine if a biomarker for
the target bacterium is present. For example, MALDI/TOF-MS analysis
can be used to produce a mass spectrum. The mass spectrum is
analyzed to determine if one or more biomarkers for the
bacteriophage are present. If such biomarkers are present, this is
an indirect indication that at least a certain concentration of the
target bacterium was present in the originally sampled mixture.
[0034] It should be appreciated that the need for the purification
step may not be necessary in situations in which the portion of the
signal associated with other biological materials can be filtered,
eliminated or otherwise ignored and/or in situations in which the
amplification step has a gain such that the portion of the mass
spectrum associated with the bacteriophage is likely to exceed any
background signal associated with other biological materials and/or
situations in which a false positive is considered to be remote.
Further, it should also be appreciated that one of the other
biological materials that can make it difficult to determine if the
biomarker for the bacteriophage is present is a wild version of the
bacteriophage that is present in the liquid solution that is being
tested. The purification step addresses the presence of a wild
bacteriophage.
[0035] If the presence or possible presence of a wild bacteriophage
in the solution or mixture being tested is not a concern, it is
also possible to perform a purification step after the
amplification step. However, in this case, the bacteriophage is
separated from the remainder of the liquid solution. The previously
noted IMS technique can be employed. However, the magnetic bead is
coated with an antibody for the bacteriophage, rather than an
antibody for the target bacterium.
[0036] It is also possible in certain cases to eliminate the need
for a purification step by utilizing a highly specific analysis
technique, such as MS/MS, in which the biomarker for the
bacteriophage is so highly specific to the bacteriophage that there
is little likelihood of a false positive. Currently, the use of
MS/MS is limited to capsid proteins that are less than 7000 Da.
[0037] With reference to FIG. 1, the immunomagnetic separation
(IMS) technique for capturing the target bacterium in a mixture and
separating any captured target bacterium from other biological
material in the mixture is described. Microsize beads are
constructed from an iron oxide core coated with a polymeric
surface. Secondary antibodies raised against the Fc region of the
primary antibodies are covalently attached via a linker to the
polymer surface. The primary antibody (raised against a targeted
microorganism) is attached to the beads by strong noncovalent
interactions with the secondary antibody, which holds the primary
antibody in the proper orientation for reaction with the targeted
antigen.
[0038] With reference to FIG. 2, the immunomagnetic beads are added
to the bacterial or biological mixture that is the subject of the
analysis and incubated for 20 minutes at room temperature. The
beads are then isolated to the side of the reaction tube using a
magnet. This process allows the extraneous (non-targeted) material
to be removed by aspiration. At this stage, the beads can be washed
several times prior to re-suspending them in PBS.
[0039] With reference to FIG. 3, the bead-bacterial target complex
is admixed with a low titer suspension of bacteriophage specific to
the targeted bacterium. The titer is held low so that the mass
spectrometry signal from the virus is non-detectable. After a
40-minute incubation, the bacteriophage have completed a
propagation cycle of attachment, insertion, self-assembly, and cell
lysis resulting in the production of many progeny that are released
into the reaction milieu. The milieu is then analyzed to determine
if a biomarker for the bacteriophage is present that indirectly
indicates that the target bacterium was present. For instance, the
milieu can be analyzed by MALDI-TOF-MS using a sandwich sample
preparation technique with a ferulic acid matrix. Other MALDI
matrices known in the art are also feasible. The resulting mass
spectrum shows the presence of the bacteriophage capsid protein,
which would not have been present if the target bacterium was not
also present.
[0040] An IMS technique for capturing target bacterium in a mixture
and separating any captured target bacterium from other biological
material in the mixture and subsequent MALDI-TOF/MS analysis are
described in U.S. patent application Ser. No. 10/063,346, entitled
"Method for Determining if a Type of Bacteria is Present in a
Mixture," filed on Apr. 12, 2002, which is incorporated herein, in
its entirety, by reference.
EXAMPLE
[0041] As described hereinafter, an embodiment of the method has
been used to reduce the detection limit for E. coli to less than
5.0.times.10.sup.4 cells mL.sup.-1. The method used immunomagnetic
beads coated with antibodies against E. coli, hereinafter referred
to as the target-bead complex, to isolate the bacterium from
solution. The target-bead complex was then re-suspended in a
solution containing MS2, a bacteriophage that is specific for E.
coli. The MS2 bacteriophage concentration was adjusted so that the
ion signal from the capsid protein of the MS2 bacteriophage was
below the detection limit of the mass spectrometer. After a
40-minute incubation period, an aliquot of the solution was removed
and analyzed by the on-probe MALDI-TOF-MS procedure for the 13 kDa
capsid protein. The [M+H].sup.+ (m/z 13,726) and [M+2H].sup.+2 (m/z
6865) ion signals for the MS2 capsid protein were detected (FIG.
4).
[0042] With reference to FIG. 5A, application of the process to a
mixture that contains a concentration of 5.0.times.10.sup.6 E. coli
cells mL.sup.-1 yields a mass spectrum with protein signals for
both E. coli and the MS2 bacteriophage. The process was repeated
for decreasing concentrations of E. coli. Specifically, with
reference to FIG. 5B, the process was repeated for a concentration
of .about.5.0.times.10.sup.5 E. coli cells mL.sup.-1. In this case,
the mass spectrum fails to definitively show any protein signals
for the E. coli cells, but does show the protein signals for the
MS2 bacteriophage capsid protein. With reference to FIG. 5C, the
process was repeated for an E. coli concentration of
.about.5.0.times.10.sup.4 cells mL.sup.-1. In this case, the mass
spectrum fails to definitively show any protein signals for the E.
coli cells but still show protein signals for the MS2 bacteriophage
capsid protein. These results indicate that E. coli was trapped by
the immunomagnetic beads and then infected by the MS2 virus, which
was able to multiply and increase the concentration of the capsid
protein to a detectable level. Presently, target bacterium
concentrations of as low as .about.1.0.times.10.sup.3 cells
mL.sup.-1 have been indirectly detected using this process.
[0043] The following describes various aspects of the embodiment of
the method implemented with respect to the example of the detection
of E. coli.
[0044] E. coli Preparation
[0045] The E. coli bacteria were grown in trypticase soy broth
(TSB) (Difco, Detroit, Mich.) with incubation at 37.degree. C.
using standard microbial methods.
[0046] Bacteriophage Propagation
[0047] Bacteriophage propagation was performed in accordance to the
Adams agar-overlay method as described in M. H. Adams'
Bacteriophages (Interscience Publishers, Inc., New York, 1959).
Briefly, a soft-agar/host covering was prepared by overlaying agar
plates (trypticase soy agar, Difco) with a 2.5 mL of melted 0.5%
agar (same medium), which contained two drops of a 20 hr host in
TSB. The soft-agar covering was allowed to harden before the
addition of a 0.5 mL overlay of a concentrated suspension of MS2 ,
prepared by re-hydrating freeze-dried MS2 in TSB. After 24 hours,
the soft agar was scraped off the surface of the agar plates and
centrifuged (1000 G) for 25 min to sediment the cellular debris and
agar. The supernatant was conserved, passed through 0.22 .mu.m
Millipore filters, and stored by refrigeration at 4-8.degree.
C.
[0048] Immunomagnetic Bead Preparation
[0049] Rabbit anti-E. coli IgG antibodies (Cortex Biochem, San
Leandro, Calif.) were attached to the immunomagnetic beads
(MagaBeads, Goat anti-Rabbit IgG F(c), Cortex Biochem) using the
manufacturer's suggested protocol.
[0050] Immunomagnetic separation (IMS) E. coli
[0051] Escherichia coli were isolated from aqueous suspensions by
affinity capture using the immunomagnetic beads. Suspensions of
bacteria were prepared in 1.5 mL microcentrifuge tubes (Brinkmann
Instruments, Inc., Westbury, N.Y.) by combining 100 .mu.L of broth
media with 900 .mu.L of phosphate buffer saline (PBS, 0.01M
Na.sub.2HPO.sub.4, 0.15M NaCl titrated to pH 7.35 with HCl). Cell
concentrations were determined using a Petroff-Hauser counting
chamber (Hauser Scientific, Horsham, Pa.).
[0052] The immunomagnetic separation (IMS) procedure developed in
this investigation involved the following steps: In the first step,
a 30 .mu.L aliquot of the immunomagnetic beads were added to the
bacterial sample solution and incubated for 20 minutes at room
temperature with continuous shaking. The second step involved
concentrating the beads to the side of the sample tube using a
magnetic particle concentrator (Dynal, Lake Success, N.Y.) and
removing the supernatant using a 1 mL pipette. In the third step,
the magnet was removed and the beads were re-suspended in 1 mL of
fresh PBS with vigorous shaking for 20 sec to wash away any
nonspecifically adhering components. The bead suspension was then
transferred to a new tube and steps 2 and 3 repeated one more time.
In the fourth and final step, the beads were isolated with the
magnet followed by decanting the buffer wash to waste and
re-suspending the beads in 500 .mu.L of deionized water.
Subsequently, the bead-E. coli complexes were admixed with a low
titer (below the detection limit of the mass spectrometer) of the
MS2 bacteriophage and incubated at room temperature with gentle
shaking for 40 minutes. An aliquot of the suspension was then
removed and analyzed for the MS2 capsid protein using a sandwich
sample preparation with a ferulic acid matrix (12.5 mg of ferulic
acid in 1 mL of 17% formic acid: 33% acetonitrile: 50% deionized
H.sub.2O).
[0053] MALDI-TOF MS
[0054] All mass spectra were generated on a Voyager-DE STR+ (AB
Applied Biosystems, Framingham, Mass.) MALDI-TOF mass spectrometer,
operating in the positive linear mode. The following parameters
were used: accelerating voltage 25 kV, grid voltage 92% of
accelerating voltage, extraction delay time of 350 nsec, and low
mass ion gate set to 4 kDa. The laser intensity (N.sub.2, 337 nm)
was set just above the ion generation threshold and pulsed every
300 ns. Mass spectra were acquired from each sample by accumulating
100 laser shots from five different sample spots (final
spectrum=average of 5.times.100 laser shots).
[0055] It should be appreciated that to assure that the detection
of a biomarker for the bacteriophage indicates that the target
bacterium is present in the liquid solution, a concentration of
"parent" bacteriophage is applied to the liquid solution that is
below the detection limit for the bacteriophage or biomarker for
the bacteriophage for whatever analysis technique is employed. This
assures that if the bacteriophage or the biomarker for the
bacteriophage is detected by the analysis technique, the detectable
concentration of the bacteriophage or biomarker is attributable to
progeny bacteriophage, i.e., bacteriophage resulting from the
replication of the bacteriophage by the target bacterium present in
the liquid solution. In certain situations, the use of such a
concentration of "parent" bacteriophage has a multiplicity of
infection ("MOI") (i.e., the ratio of the number of parent
bacteriophage to the number of target bacterium) that is too low to
produce a sufficient concentration of bacteriophages or biomarkers
for the bacteriophage for detection.
[0056] To overcome the drawbacks associated with a low MOI, a
sufficiently high concentration of "parent" bacteriophage is added
to the liquid solution. In this case, the concentration of the
"parent" bacteriophage added to the solution may exceed the
detection limit of whatever analysis technique is employed to
detect the bacteriophage or biomarker of the bacteriophage.
Consequently, analysis of a liquid solution treated in this manner
could detect the "parent" bacteriophages that were added to the
solution, rather than the progeny bacteriophage resulting from
replication by the target bacteria.
[0057] Consequently, another embodiment of the process applies a
concentration of "parent" bacteriophage to the solution that is
capable of being distinguished from any progeny bacteriophage. In
one embodiment, the parent bacteriophage (i.e., the bacteriophage
initially applied to the solution) are "tagged" so that whatever
analysis technique is employed is inherently capable of
distinguishing the parent bacteriophage or parent bacteriophage
biomarkers from the progeny bacteriophage or biomarkers for the
progency bacteriophage. For example, if a mass spectral analysis
technique is employed, the parent bacteriophage are "tagged" with a
substance that alters or shifts the mass spectrum of the parent
bacteriophage relative to the progeny bacteriophage, which will not
inherit the "tag" from the parent bacteriophage. For example, a
biotinylated bacteriophage is employed as a parent bacteriophage
and has a different mass spectrum than the progeny bacteriophage
produced by the biotinylated bacteriophage infecting target
bacterium present in the solution. Other "tags" can be employed for
other types of analytical techniques.
[0058] In another embodiment, the parent bacteriophage possesses a
characteristic that allows the parent bacteriophage to be separated
from any of the progeny bacteriophage in the liquid solution prior
to analysis, thereby assuring that most, if not all of the
bacteriophage present in the liquid solution after separation are
progeny bacteriophage resulting from the replication of the parent
bacteriophage by target bacteria present in the liquid solution. In
one embodiment, biotinylated bacteriophage are initially applied to
the liquid solution. Biotinylated bacteriophage are highly
attracted to strepavidin. Consequently, to separate the
biotinylated bacteriophage from the liquid solution a strepavidin
probe is utilized. In one embodiment, the biotinylated
bacteriophage are attached to a strepavidin coated probe and the
probe is placed in the liquid solution. In this case, separation of
the biotinylated bacteriophage from the liquid solution after the
incubation period is accomplished by removing the probe from the
liquid solution. In another embodiment, strepavidin-coated magnetic
beads are applied to the liquid solution. The biotinylated
bacteriophages are attached to the strepavidin-coated magnetic
beads prior to the application of the beads to the solution.
Alternatively, the beads are used to pick up biotinylated
bacteriophages that were previously added to the solution and then
separated from the liquid solution using a magnet. In yet another,
embodiment a strepavidin coated probe (e.g., a slide) is applied to
the liquid solution after the incubation period. The biotinylated
bacteriophages adhere to the probe and then the probe is separated
from the liquid solution. Regardless of the manner in which the
biotinylated bacteriophages are separated from the liquid mixture,
at least a portion of the solution is then subjected to analysis to
determine if the bacteriophage or a biomarker for the bacteriophage
is present, which indirectly indicates that the target bacteria was
present in the solution.
* * * * *